Anthony Sharp

Protective effects of a peroxisome proliferator-activated receptor-β/δ agonist in experimental autoimmune encephalomyelitis

Agonists of the peroxisome proliferator-activated receptor gamma (PPARg) exert anti-inflammatory and anti-proliferative effects which led to testing of these drugs in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In contrast, the effect of PPARdelta (PPARy) agonists in EAE is not yet known. We show that oral administration of the selective PPARy agonist GW0742 reduced clinical symptoms in C57BL/6 mice that had been immunized with encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide. In contrast to previous results with PPARg agonists, GW0742 only modestly attenuated clinical symptoms when the drug was provided simultaneously with immunization, but a greater reduction was observed if administered during disease progression. Reduced clinical symptoms were accompanied by a reduction in the appearance of new cortical lesions, however cerebellar lesion load was not reduced. Treatment of T-cells with GW0742 either in vivo or in vitro did not reduce IFNg production; however GW0742 reduced astroglial and microglial inflammatory activation and IL-1h levels in EAE brain. RTPCR analysis showed that GW0742 increased expression of some myelin genes. These data demonstrate that PPARy agonists, like other PPAR ligands, can exert protective actions in an autoimmune model of demyelinating disease. D 2005 Elsevier B.V. All rights reserved.

The anti-inflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1

DMF (dimethyl fumarate) exerts anti-inflammatory and pro-metabolic effects in a variety of cell types, and a formulation (BG-12) is being evaluated for monotherapy in multiple sclerosis patients. DMF modifies glutathione (GSH) levels that can induce expression of the anti-inflammatory protein HO-1 (haem oxygenase-1). In primary astrocytes and C6 glioma cells, BG-12 dose-dependently suppressed nitrite production induced by either LI [LPS (lipopolysaccharide) at 1 μg/ml plus IFNγ (interferon γ) at 20 units/ml] or a mixture of pro-inflammatory cytokines, with greater efficacy in C6 cells. BG-12 reduced NOS2 (nitric oxide synthase 2) mRNA levels and activation of a NOS2 promoter, reduced nuclear levels of NF-κB (nuclear factor κB) p65 subunit and attenuated loss of IκBα (inhibitory κBα) in both cell types, although with greater effects in astrocytes. In astrocytes, LI decreased mRNA levels for GSHr (GSH reductase) and GCL (c-glutamylcysteine synthetase), and slightly suppressed GSHs (GSH synthetase) mRNAs. Co-treatment with BG-12 prevented those decreased and increased levels above control values. In contrast, LI reduced GSHp (GSH peroxidase) and GCL in C6 cells, and BG-12 had no effect on those levels. BG-12 increased nuclear levels of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), an inducer of GSH-related enzymes, in astrocytes but not C6 cells. In astrocytes, GSH was decreased by BG-12 at 2 h and increased at 24 h. Prior depletion of GSH using buthionine-sulfoximine increased the ability of BG-12 to reduce nitrites. In astrocytes, BG-12 increased HO-1 mRNA levels and effects on nitrite levels were blocked by an HO-1 inhibitor. These results demonstrate that BG-12 suppresses inflammatory activation in astrocytes and C6 glioma cells, but with distinct mechanisms, different dependence on GSH and different effects on transcription factor activation.

The heat-shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin suppresses glial inflammatory responses and ameliorates experimental autoimmune encephalomyelitis

The heat‐shock response (HSR), a highly conserved cellular response, is characterized by rapid expression of heat‐shock proteins (HSPs), and inhibition of other synthetic activities. The HSR can attenuate inflammatory responses, via suppression of transcription factor activation. A HSR can be induced pharmacologically by HSP90 inhibitors, through activation of the transcription factor Heat Shock Factor 1 (HSF1). In the present study we characterized the effects of 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG), a less toxic derivative of the naturally occurring HSP90 inhibitor geldanamycin, on glial inflammatory responses and the development of experimental autoimmune encephalomyelitis. In primary enriched glial cultures, 17‐AAG dose dependently reduced lipopolysaccharide‐dependent expression and activity of inducible nitric oxide synthase, attenuated interleukin (IL)‐1β expression and release, increased inhibitor of κB protein levels, and induced HSP70 expression. 17‐AAG administration to mice immunized with myelin oligodendrocyte glycoprotein peptide prevented disease onset when given at an early time, and reduced clinical symptoms when given during ongoing disease. T cells from treated mice showed a reduced response to immunogen re‐stimulation, and 17‐AAG reduced CD3‐ and CD28‐dependent IL‐2 production. Together, these data suggest that HSP90 inhibitors could represent a new approach for therapeutic intervention in autoimmune diseases such as multiple sclerosis.

The Heat Shock Response Inhibits NF-κB Activation, Nitric Oxide Synthase Type 2 Expression, and Macrophage/Microglial Activation in Brain

The heat shock response (HSR) provides protection against stress-induced damage, and also prevents initiation of inflammatory gene expression via inhibition of NFκB activation. This article describes experiments demonstrating that the HSR prevents induction of nitric oxide synthase type 2 (NOS2) in rat brain. Twenty four hours after intrastriatal injection of lipopolysaccharide (LPS), IL-1β, and IFN-γ, NOS2 immunoreactive cells were detected in striatum, corpus callosum, and to a lesser extent in cortex. Induction of a HSR by whole body warming to 41°C for 20 minutes, done 1 day before LPS plus cytokine injection, reduced the number of NOS2-positive staining cells to background levels. Staining for ED1 antigen revealed that the HSR also suppressed microglial/brain macrophage activation in the same areas. Striatal injection of LPS and cytokines induced the rapid activation of NFκB, and this activation was prevented by prior HS, which also increased brain IκB-α expression. These results suggest that establishment of a HSR can reduce inflammatory gene expression in brain, mediated by inhibition of NFκB activation, and may therefore offer a novel approach to treatment and prevention of neurological disease and trauma.

Increasing CNS Noradrenaline Reduces EAE Severity

The endogenous neurotransmitter noradrenaline (NA) is known to exert potent anti-inflammatory effects in glial cells, as well as provide neuroprotection against excitatory and inflammatory stimuli. These properties raise the possibility that increasing levels of NA in the central nervous system (CNS) could provide benefit in neurological diseases and conditions containing an inflammatory component. In the current study, we tested this possibility by examining the consequences of selectively modulating CNS NA levels on the development of clinical signs in experimental autoimmune encephalomyelitis (EAE). In mice immunized with myelin oligodendrocyte glycoprotein peptide to develop a chronic disease, pretreatment to selectively deplete CNS NA levels exacerbated clinical scores. Elevation of NA levels using the selective NA reuptake inhibitor atomoxetine did not affect clinical scores, while treatment of immunized mice with the synthetic NA precursor l-threo-3,4-dihydroxyphenylserine (l-DOPS) prevented further worsening. In contrast, treatment of mice with a combination of atomoxetine and l-DOPS led to significant improvement in clinical scores as compared to the control group. The combined treatment reduced astrocyte activation in the molecular layer of the cerebellum as assessed by staining for glial fibrillary protein but did not affect Th1 or Th17 type cytokine production from splenic T cells. These data suggest that selective elevation of CNS NA levels could provide benefit in EAE and multiple sclerosis without influencing peripheral immune responses.

Suppressive effects of ansamycins on inducible nitric oxide synthase expression and the development of experimental autoimmune encephalomyelitis

The production of nitric oxide by the inflammatory isoform of nitric oxide synthase (NOS2) in brain glial cells is thought to contribute to the causes and development of neurological diseases and trauma. We previously demonstrated that activation of a heat shock response (HSR) by hyperthermia reduced NOS2 expression in vitro, and in vivo attenuated the clinical and histological symptoms of the demyelinating disease experimental autoimmune encephalomyelitis (EAE; Heneka et al. [2001] J. Neurochem. 77:568–579). Benzoquinoid ansamycins are fungal‐derived antibiotics with tyrosine kinase inhibitory properties, and which also induce a HSR by allowing activation of HS transcription factor HSF1. We now show that two members of this class of drugs (geldanamycin and 17‐allylamino‐17‐demethoxygeldanamycin) also induce a HSR in primary rat astrocytes and rat C6 glioma cells. Both drugs dose‐dependently reduced nitrite accumulation, NOS2 steady‐state mRNA levels, and the cytokine‐dependent activation of a rat 2.2‐kB NOS2 promoter construct stably expressed in C6 cells. These inhibitory effects were partially reversed by quercetin, a bioflavonoid which prevents HSF1 binding to DNA and thus attenuates the HSR. Ansamycins increased mRNA levels of the inhibitory IκBα protein, suggesting that inhibition of NFκB activation could contribute to their suppressive effects. Finally, in C57BL/6 mice actively immunized to develop EAE, a single injection of geldanamycin at 3 days after immunization reduced disease onset by over 50%. These results indicate that ansamycins can exert potent anti‐inflammatory effects on brain glial cells which may provide therapeutic benefit in neuroinflammatory diseases.

Inhibitory and Stimulatory Effects of Lactacystin on Expression of Nitric Oxide Synthase Type 2 in Brain Glial Cells THE ROLE OF IκB-β

Expression of inflammatory nitric oxide synthase (NOS2) is mediated by transcription factor NFκB. By using the specific proteasome inhibitor lactacystin to examine IκB degradation, we observed a paradoxical increase in lipopolysaccharide- and cytokine-dependent NOS2 expression at low concentrations or when lactacystin was added subsequent to cytokines. Lactacystin reduced the initial accumulation of NOS2 mRNA but reduced its subsequent decrease. Lactacystin increased NOS2 promoter activation after 24 h, but not after 4 h, and similarly prevented initial NFκB activation and at later times caused NFκB reactivation. Lactacystin reduced initial degradation of IκB-α and IκB-β, however, at later times selectively increased IκB-β, which was predominantly non-phosphorylated. Expression of full-length rat IκB-β, but not a carboxyl-terminal truncated form, inhibited NOS2 induction and potentiation by lactacystin. Lactacystin increased IκB-β expression in the absence of NOS2 inducers, as well as expression of heat shock protein 70, and the heat shock response due to hyperthermia increased IκB-β expression. These results suggest that IκB-β contributes to persistent NFκB activation and NOS2 expression in glial cells, that IκB-β is a stress protein inducible by hyperthermia or proteasome inhibitors, and that delayed addition of proteasome inhibitors can have stimulatory rather than inhibitory actions.

Rapamycin reduces clinical signs and neuropathic pain in a chronic model of experimental autoimmune encephalomyelitis.

Current treatments used in Multiple Sclerosis (MS) are partly effective in the early stages of the disease but display very limited benefits in patients affected by progressive MS. One possible explanation is that these therapies are unable to target the inflammatory component most active during the progressive phase of the disease, and compartmentalized behind the blood-brain barrier. Our findings show that Rapamycin ameliorates clinical and histological signs of chronic EAE when administered during ongoing disease. Moreover, Rapamycin significantly reduced the hyperalgesia observed before clinical development of EAE which, in turn, is completely abolished by the administration of the drug.

The heat shock response reduces myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis in mice

The stress response (SR) can block inflammatory gene expression by preventing activation of transcription factor nuclear factor‐kappa B (NF‐κB). As inflammatory gene expression contributes to the pathogenesis of demyelinating diseases, we tested the effects of the SR on the progression of the demyelinating disease experimental autoimmune encephalomyelitis (EAE). EAE was actively induced in C57BL/6 mice using an encephalitogenic myelin oligodendrocyte glycoprotein (MOG35−55) peptide. Whole body hyperthermia was used to induce a heat shock response (HSR) in immunized mice 2 days after the booster MOG35−55 peptide injection. The HSR reduced the incidence of EAE by 70%, delayed disease onset by 6 days, and attenuated disease severity. The HSR attenuated leukocyte infiltration into CNS assessed by quantitation of perivascular infiltrates, and by reduced staining for CD4 and CD25 immunopositive T‐cells. T‐cell activation, assessed by the production of interferon γ (IFNγ) in response to MOG35−55, was also decreased by the HSR. The HSR reduced inflammatory gene expression in the brain that normally occurs during EAE, including the early increase in RANTES (regulated on activation of normal T‐cell expressed and secreted) expression, and the later expression of the inducible form of nitric oxide synthase. The early activation of transcription factor NF‐κB was also blocked by the HSR. The finding that the SR reduces inflammation in the brain and the clinical severity of EAE opens a novel therapeutic approach for prevention of autoimmune diseases.

Local anesthetics potentiate nitric oxide synthase type 2 expression in rat glial cells.

Expression of the calcium-independent nitric oxide synthase (NOS2) contributes to damage in neurologic disease and trauma. The effects of local anesthetics on NOS2 expression have not been examined. The authors tested the effects of four local anesthetics on the expression of NOS2 in immunostimulated rat C6 glioma cells. Incubation with local anesthetics alone did not induce nitrite accumulation; however, the nitrite production induced by stimulation with bacterial endotoxin lipopolysaccharide (LPS) and interferon-&ggr; (IFN-&ggr;) was increased in a dose-dependent manner by bupivacaine (maximal 3-fold at 360 &mgr;M), tetracaine (maximal 7-fold at 360 &mgr;M), and lidocaine at higher doses (5-fold increase at 3.3 mM). Significant increases in nitrite production were observed in concentrations of bupivacaine or tetracaine as low as 120 &mgr;M, which correspond to 30 &mgr;g/mL (.003% weight/volume). In contrast, ropivacaine had little effect on nitrite production (160% of control values) and only at the highest concentration (3.3 mM, corresponding to 890 &mgr;g/mL or 0.089% w/v) tested. Increased nitrite production was not caused by cytotoxic effects of the drugs used, as assessed by release of intracellular lactate dehydrogenase. Increased nitrite production was accompanied by increased NOS2 catalytic activity, steady state mRNA levels, and promoter activation. These results demonstrate that submillimolar doses of two commonly used local anesthetics can increase glial NOS2 expression.